The Genome and Transcriptome of the Zoonotic Hookworm Ancylostoma Ceylanicum Identify Infection-Specific Gene Families

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The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families

Erich M Schwarz1, Yan Hu2,3, Igor Antoshechkin4, Melanie M Miller3, Paul W Sternberg4,5 & Raffi V Aroian2,3

Hookworms infect over 400 million people, stunting and closely related to the free-living Caenorhabditis elegans than is the impoverishing them1–3. Sequencing hookworm genomes and free-living Pristionchus pacificus (Fig. 2)12–15. Treatments effective finding which genes they express during infection should against A. ceylanicum might thus also prove useful against other help in devising new drugs or vaccines against hookworms4,5. strongylids, such as Haemonchus contortus, that infect farm ani- Unlike other hookworms, Ancylostoma ceylanicum infects mals and depress agricultural productivity16. Characterizing the both humans and other mammals, providing a laboratory genome and transcriptome of A. ceylanicum is a key step toward such model for hookworm disease6,7. We determined an comparative analysis. A. ceylanicum genome sequence of 313 Mb, with We assembled an initial A. ceylanicum genome sequence of 313 Mb transcriptomic data throughout infection showing expression and a scaffold N50 of 668 kb, estimated to cover ~95% of the genome, of 30,738 genes. Approximately 900 genes were upregulated with Illumina sequencing and RNA scaffolding17,18 (Supplementary during early infection in vivo, including ASPRs, a cryptic Tables 1–3). The genome size was comparable to those of Ancylostoma subfamily of activation-associated secreted proteins (ASPs)8. caninum (347 Mb)19 and H. contortus (320–370 Mb)20,21 but larger Genes downregulated during early infection included than those of N. americanus, C. elegans and P. pacificus (100– ion channels and G protein–coupled receptors; this 244 Mb)22–24. We found that 40.5% of the genomic DNA was repeti- downregulation was observed in both parasitic and free-living tive, twice as much as in N. americanus, C. elegans or P. pacificus nematodes. Later, at the onset of heavy blood feeding, C-lectin (17–24%). We predicted 26,966 protein-coding genes25 with products genes were upregulated along with genes for secreted clade V of ≥100 residues (Supplementary Table 4). We also predicted 10,050 proteins (SCVPs), encoding a previously undescribed protein genes with products of 30–99 residues, to uncover smaller proteins family. These findings provide new drug and vaccine targets that might aid in parasitism26. With RNA sequencing (RNA-seq), and should help elucidate hookworm pathogenesis. we detected expression of 23,855 (88.5%) and 6,883 (68.5%) of these genes, respectively (Fig. 3). The two hookworm species causing the most infections are Necator The genomes of plant-parasitic, necromenic and animal-parasitic americanus and Ancylostoma duodenale, which are generally restricted nematodes have all acquired bacterial genes through horizontal gene to human hosts1,9. Hookworms are free living during part of their transfer (HGT)27,28. We detected one instance of bacterial HGT in life cycle, with eggs hatching in soil and larvae feeding on bacteria A. ceylanicum: Acey_s0012.g1873, a homolog of the N-acetylmuramoyl- through the first and second larval stages. At the infectious third- l-alanine amidase amiD, which encodes a protein that may help stage larval phase (L3i), hookworms cease feeding and wait until they bacteria recycle their murein29. Acey_s0012.g1873 was strongly encounter a human host. They generally enter their host by burrow- expressed in L3i and then downregulated in all later stages of ing into skin, although Ancylostoma can alternatively enter by being infection. It has nine predicted introns, presumably acquired after swallowed. Hookworms then pass through the bloodstream, lungs HGT; it has only one homolog in the entire nematode phylum and digestive tract to the small intestine, where they affix themselves, (NECAME_15163 from N. americanus) but many bacterial homologs mature to adulthood, mate and lay eggs that are excreted by the (Supplementary Fig. 1 and Supplementary Table 5). The sap-feeding host1. The ability to culture A. ceylanicum in golden hamster allows insects Acyrthosiphon pisum and Planococcus citri also have amiD it to be used as a model system for the human-specific hookworms genes, acquired by HGT, that may promote bacterial lysis30,31. N. americanus and A. duodenale, upon which new drug and vaccine To find genes acting at specific points of infection, we carried out candidates can be tested (Fig. 1)6,10,11. Human-specific hookworms RNA-seq on specimens collected at developmental stages spanning belong to a class of parasitic nematodes, strongylids, that are more the onset and establishment of infection by A. ceylanicum in golden

1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. 2Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. 3Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, USA. 4Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA. 5Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA. Correspondence should be addressed to E.M.S. ([email protected])

Received 12 January 2014; accepted 5 February 2015; published online 2 March 2015; doi:10.1038/ng.3237

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Figure 1 Life cycle of A. ceylanicum. A. ceylanicum hatch in feces and 19.D L3i 24.HCM grow as free-living first- to third-stage (L1–L3) larvae. Before exiting the third larval stage, they mature into infectious third-stage (L3i) larvae, arresting further development until they are inside a host. In 24 h after gavage into golden hamsters, A. ceylanicum are still in the stomach but 17.D 24.PI have exited the L3i stage (24.PI). A standard model for parasite infection is to incubate L3i larvae for 24 h in hookworm culture medium (24.HCM), which evokes changes in larval shape and behavior thought to mimic those of 24.PI larvae in vivo. By 5 d after infection, larvae have migrated further to the intestine, affixed themselves there and grown into early fourth-stage 12.D 5.D (L4) larvae (5.D; female shown) with visible sexual differentiation. By 12 d (12.D; female shown), they start heavy blood feeding and become young adults with mature males and a few gravid females, with little or no egg laying. By 17 d (17.D; male shown), they are fully mature adults. They begin laying many eggs that are deposited outside the host during defecation, renewing the life cycle. From 19 d onward (19.D; male shown), they remain fertile adults for weeks in hamsters. Scale bars: with N. americanus or other nematodes might encode previously 100 m (L3i through 5.D), 500 m (12.D) and 1 mm (17.D and 19.D). µ µ undescribed components of infection (Supplementary Table 10). Proteases, protease inhibitors, nucleases and protein synthesis were hamster (Figs. 1 and 3, and Supplementary Table 6), beginning at upregulated during early infection (L3i to 24.PI; Supplementary L3i and followed by 24 h either of incubation in hookworm culture Tables 9a and 11a); proteases and protease inhibitors were medium (24.HCM), a standard model for early hookworm infection32, also upregulated after L3i in N. americanus24, as were proteases in or infection in the hamster stomach (24.PI). We found 942 genes to H. contortus21. Secreted proteases could allow hookworms to digest be significantly upregulated from L3i after 24 h of infection in vivo host proteins in blood and intestinal mucosa6,11,35–37. Secreted (Supplementary Table 7). In contrast, we observed only 240 genes proteases might also digest and inactivate proteins of the host’s significantly upregulated from L3i after 24 h of incubation in HCM, immune system37,38. Conversely, secreted protease inhibitors could of which 141 were also upregulated with in vivo infection. This lower also suppress host immunity39–41. number matches previous observations32 and shows that infection G protein–coupled receptors (GPCRs), receptor-gated ion channels in vivo has stronger effects on gene activity than its in vitro model. and neurotransmission-related functions in general were downregu- We linked known or probable gene functions to steps of infec- lated during early infection (L3i to 24.PI), along with transcription tion by assigning gene ontology (GO) terms to A. ceylanicum genes33 factors (Supplementary Tables 9b and 11b). We observed the and computing which GO terms were over-represented among same pattern among genes downregulated in the transition from L3 genes upregulated or downregulated in developmental transitions to fourth-stage (L4) larvae both in H. contortus21 and C. elegans42 (Supplementary Tables 8 and 9)34. We also analyzed homologous (Supplementary Table 8). This finding is consistent with down- gene families for disproportionate upregulation or downregulation; regulation after L3 of sensory perception and transcription genes in in particular, gene families identified by orthology of A. ceylanicum both C. elegans43 and N. americanus24 and of ion channel genes in A. caninum and Brugia malayi32,44. Such downregulation might thus A. ceylanicum C. elegans be conserved in both parasitic and free-living nematodes. A. ceylanicum 9,860 7,042 Among gene families upregulated during early infection, we found

N. americanus 7,506 6,225 some already known from other parasitic nematodes, such as ASPs

H. contortus 5,268 4,576

C. elegans 7,042 11,323 L3i 24.HCM 24.PI 5.D 12.D 17.D 19.D

C. briggsae 6,933 10,795

P. pacificus 4,833 4,979

B. xylophilus 5,373 5,587

M. hapla 4,161 4,376

A. suum 5,955 6,209

B. malayi 5,312 5,545

D. immitis 5,166 5,308

T. spiralis 3,015 3,186

Figure 2 Evolutionary relatedness of A. ceylanicum to other nematodes. 13 14 The phylogeny is derived from van Megen et al. and Kiontke et al. . log2 (TPM) 0 15 0.5 1.0 1.5 2.0 2.5 3.0 N. americanus and H. contortus are strongylid parasites and the closest –2.5 –2.0 –1.5 –1.0 –0.5 ≥ ≤–3.0 relatives of A. ceylanicum. C. elegans, C. briggsae and P. pacificus are free-living, non-parasitic nematodes. Nematodes from distinct groups Figure 3 RNA expression levels for 30,738 A. ceylanicum genes. 12 (clades) within the phylum are color-coded: black, A. ceylanicum and Gene activity during infection is shown in log2-transformed transcripts close relatives, clade V; green, plant parasites, clade IV; pink, ascarid and per million (TPM), with k partitioning of the genes into 20 groups. filarial animal parasites, clade III; orange, Trichinella, an animal parasite Genes in yellow and blue are up- and downregulated, respectively; TPM from clade I. To the right are the numbers of strictly orthologous genes for values are shown ranging from ≤2−3 to ≥23. Developmental stages are as A. ceylanicum or C. elegans and other species. Self-comparisons (bold) in Figure 1. Changes in gene expression after 24 h of growth in HCM list all strictly defined orthologs within a genome. A. ceylanicum and (24.HCM) are relatively minor, as opposed to the far-reaching changes C. elegans have similar orthology to diverse nematode species. in gene expression seen after 24 h of infection in vivo (24.PI).

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(Supplementary Table 12a)21,24,45,46. ASP genes encode a diverse set in some strongylids (for example, N. americanus; Supplementary of secreted cysteine-rich proteins, whose functions probably include Tables 13 and 14) but not all (for example, H. contortus). Most ASPR blocking immune responses and blood clotting8. However, we also proteins were predicted to be secreted (Supplementary Table 4), and found a family of 92 genes collectively upregulated during early infec- one ASPR in Heligmosomoides bakeri is secreted by parasitic adults46. tion in vivo (24.PI; q value = 0.003) that had no obvious similarity Thus, like ASPs, ASPRs might comprise an important element of to known gene families (Supplementary Tables 4 and 12a). By con- hookworm infection in vivo. trast, upregulation of these genes after 24 h of simulated infection A. ceylanicum had 432 ASP genes, noticeably more than the related in vitro was insignificant (24.HCM; q value = 0.93). These homologs parasites N. americanus (128 genes) and H. contortus (161 genes) and were distantly related to ASPs, so we termed them ASP-related genes remarkably more than the non-parasitic C. elegans and P. pacificus (ASPRs; Fig. 4 and Supplementary Fig. 2). We found other ASPRs (35 and 33 genes, respectively). A. ceylanicum and N. americanus

ASPRs

A H

S 2

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0 / /

/ 0 1-169 Nam-ASP /5-13 ASPR-s00 9 - /30-149 ASPR-s0 0 9 9 3 4 1 P ASPR-s01 S 2 -140 4 9/13-152 s /7-143 0

1 0 3-137 6 0 5 4 7 4 5 R ASPR-s 0 P ASPR-s0623.g780/17-15 4 7 ASPR-s059 1 5 8 /7-15 8 7 7 42/23 7 R-g17038/54- 7 9-236 0 -17 - R 3 031.g2245/ 0 . 1

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g -14 g

- 1 Nam-ASPR-g g 19/54- 42.g572/ g 1

. s 1 2 3

2 1 .

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1 ASPR-s00 ASPR-s003 - -129 15.g2843/69-19 p ASPR-s0352.g3266/ 8 5/8-90 5 .

8 0 7

8 7 -243 3396/7 28/9 8 15528/5- g

R 8

- R 1 1426/8-14 2

7 ASP-s1162.g3713/1 7 5 5 Nam-ASPR-g13006 1 1 0 5 7 11/40 711/1-14

0 022.g49 268/2-153 P Oden-AS R-g047 P 8 .g2468/26-1 4 4 /15

0 4 6 0064.g 8 18.g3621/62- 0.g90 .g2249 4 1 77/1-144 ASPR- 0 Nam-ASPR- 06.g37 457.g1813/ S

/ 0 S 0 0 30/4-164 / / / 2 g1889/19-146 9 s 1 .g14 2 1 s SPR

/ s

- .g208 A 415/41-1 /

8 8/129-205 2 248/6-8 - - 1 3 - 2 0 81.g1435/23-13 - -s0457.g179 3 -s0457.g1806 ASPR-s02ASPR-s00 /8-146 /1-14

- R 9.g480 8-18 - - - n SPR-254 - R n 3.g13 ASPR-s0 - R -22809/18 1417 1-16 9 2 1

1 21-146 1 80/1-11 1 73.g2087/6-14 Nam-ASPR-g0 1

P 6-155 e 3/63-19 e P

2 3 P 473 Nam-ASPR-g09 67-183 0 s0081.g1431/1 5 05.g2681/44- 6 3511/ 135 5 Nam-ASPR-g09 53/2- d 150 18 d s0015.g286 S /4-7 R-s0020.g78/37-124 -101 135 6/44-192 3 3 9-19 S 34/66- 6 g483/50- S 0 6.g 9.g111/7- 2 7 2 5 1 PR-24659 17186/88-17 A ASPR-s023 O 4

O ASPR-s0457.g1807 A A 4 ASPR-s0457.g1800/4-14 ASPR 9 ASPR ASPR-s0 Oden-A 456/2 ASP-s0010.g1 Nam-ASPR-g16545 34-15 13 0 ASPR-s0042.g574/8-15 /31-16 ASPR-s0045.g1247/8ASPR-s0287.g1455 ASP-s 9 7/7- 5 28 ASPR-s0042.g717 144 ASP 7.g2983/21-16 1 ASP-s0 102019/ 90-22 9 Oden-A ASPR-s0357.g ASPR-s0081.g 6 043.g842/1 228 3 ASPR- 10.g2 42.g709/2-159 172 ASPR-s00 ASP-s0010.g ASPR-s0081.g1427/9-146 015.g2879 0/9-1 ASP-s0010 ASPR-s0081 ASPR-s0 3 ASPR-s04 13 ASPR-s028 .g111 ASP-s06 37-115 6 0010.g1059/15-14 13 1 7 Oden-ASPR /10-141 ASPR-s0081.g1423/27-10 -9 0 ASPR-s0046.g1420/75-15 37/9-129/13-141 ASP-s0181. 0/10-15 /3-145 ASPR-s0046.g1 4.g3130/ 09-24 ASPR- 121/20- 0 ASP-s001 010.g105 9692/58 178 8 ASPR-s004 8 3.g3068 8 ASPR-s0659.g1258/48-14 9-116 9 ASP-s000 0 0 ASPR-s0659.g1260/51-16 0-127 694/48 0.67 0.82 ASPR-s0034.g2844/49-1 /94-16 0.96

2 693/9-9 . ASPR-s0015.g28 ASP-s00 R-s0020.g73/24 9 ASPR-s0 6 ASPR-s009 -13 3 0.91 /113-19 063/47-140 . ASP-s0045.ASP-s0045.g11 1-147 0.74 4 ASPR-s0258.g R-s0123 7 ASPR-s0020.g41/19-103 36.g940/84-1.g10 0 0.58 s0002.g551/ 0.94 R-s023 2 0 0.84 Nam-A 3 ASPR-s0020.g74/8 6 0.79 1056/9-14 0. -142 ASP 0 ASP-s004 /6-150 6 -182 0. ASPR-s0031.g2246/24-100 00 0.g1054/ 7/25-165 01315/5 60/102-23 6 Nam-ASP- 6 ASP g867/6 73/63-16 4 0.18 ASP 45.g1162/15.g2 0 R-g03029 /3-14 0.33 4 ASPR-s0020.g13 ASP-s0 0 ASPR-s0025.g1272/8- R-s0010.g114 SP-g09 9 0.7 ASPR-s0188.g1150/81-182 ASP-s042 0.26 . ASP ASPR-s0148.g2676/28-103 1 0.78 0.4 399/ 0 5 Nam-A 6 0.89 ASPR-s0013.g20 g1167/ 7-15 0.96 ASP 3 5.g1157 3 0.99 1 -s0010.g107 12-1375-176 ASPR-s0013.g2039/13-14 SPR-g17814/4R-g15529/3-142 Nam-ASP-Nam-ASP 85 8 0.6 ASPR-s0020.g126/1 636.g935/ 28-14 SPR-g172 g155 509/20 59/4-169 0.28 ASPR-s0020.g128/7-136 0.48 Nam-ASP ASP-s0 8 0.93 ASP-s00 SP-g06987/22- 3-16 1 0.52 Nam-ASPR-g 8.g1294/ 17-18 0.53 0.34 0.53 0.83 0.79 ASPR-s0015.g2865 47/12 0.72 Nam-A ASP-s0046.g1373/1 6 1 ASP-s004 /3-21 0.14 0.78 Nam-A 1-30 6 067.g109/86-234 0.92 0.41 5 -g08845 0.84 1 Nam-ASP 046.g 4 0.8 69/147-195 g15941/67-216 73-165 0.84 7 Nam-ASPR-065676/24-16 -7 5 ASP-s0 0.51 1 46.g1397/3 0/42-141 -124 7 6 Nam-ASPR-g15527R-s0233.g3072/62-14 0.33 23562/12-154

7 803/10-135 ASP-s0050.g2030/ 15-80 9 0.79 Nam-ASPR-g17261/3-14

Nam-ASP- . 0.94 0.8 Nam-ASPR-g13007/14-1 SPR-12578/2-14 8 Nam-ASP- 0 ASP-s0101. 1371 0.86 0.88 7 ASP SPR-12576/2-143 ASP-s0 6.g137 -8 3 ASP-5N /3-163 0.23 ASPR-s0 9283/2 2/2-10 9 116.g6 175 ASPR-s0067.g115/31-173 0.99 0. 0.55 0.9 7 /8-18 0 0.83 Oden-ASPR-12577/2-142

1 0.99 3 . 0.92 0.81 7 Oden-A 1729 2/32-21 7-191 8 0.

. 490/13 g09588/7- 8 4 0.99 Oden-A 003.g1626 0.79 ASP- 11/9-1 0 Oden-ASPR- 1 g17854/9 6 0.79 ASP-s0007 -12 0.16 0.64 ASP-g18987/3-7-g17293/2-103 0.58 Nam-ASPR-g020 g3352/26-1 7 0.6 ASP-s0007.g3489/4-17 4 ASPR-s0012.g1 293/88-17 s0003. 4 Nam-ASP-g1 0.4 ASP-5A/2-1 1-98 82 0.84 7 ASP-s0 0.86 0.81 0.84 Nam- ASP-5B/3- 0.81 2 5 ASP-s0 134 0.31 . Nam-ASP-g ASP /348-413 8 0.92 774/6-182 -118 0 8-183 Nam-ASP 0.86 0.87 0.91 0. .g3490/37-205g1637 0.9 0428.g157.g3201/9-184 ASP-s00 0.21 0.35 Nam-ASP-g07 -s0815. 007.g3495/8- 38 0. 0.74 0.3 ASP-s 0.99 0.94 Nam-ASP-g07493/5-10 Nam-ASP 007.g3 9 ASP-s0119.g842/340-49 30-179 0.98 3 ASP-s0048 /8-178 75 0.26

174 7 ASP-s . 1 ASP-s01 0.34 ASP-s01 6 0002 02.g g249 0.68 0 ASP-s0 501/7-1 0.79 1 ASP-s0066.g3712/103-281 g2178/172/93-265 9 ASP-s0066.g3 ASP-s004 0. 0.85 0.84 9-16 -g1118.g608615/ 7/14-18 175 0.4 ASP-s0066.g3773/36-188

5 4 4 0.92 43.g241.g1600/14 0.34 0.0 ASP-s0240.g3336/ 469-612

8 0.98 ASP- 048.g1 19-1 6 . ASP-s0351.g3246/8-183

ASP-s0522.g2 /11-18 2 0.91 0 4/25-191 0. 0.94 0.48 ASP-s1508.g3901/29-138 ASP-s05 1 0.47 /79-257 s0522.g28.g1641/2 72 8 0.92 ASP-s0477. ASP-s 642/29 0.29 0.78 0128.g1456/22-100 3/224 0.94 ASP-s0477.g2 0-178 3-297 2 0.87 0.94 ASP-s1062.g3517/7-66 366/4-175 Nam-ASP-g 0.73 5 0.83 Nam-A 0522 . 22.g -384 0.52 0.98 0.37 ASP-s 0.g1034 365/1 0-45 0 2 ASP-s00 883/9-16 0.72 ASP-s0097.g2962/1 877/24-18566-42 0.89 ASP-s0048.g1657/164-324.g28 0.74 0.96 SP-g07 2887 0 0.85 ASP-s0016.g3114/ 0.71 0.99 0 0.81 ASP-s0010.g1028/4-176 ASP-s0048.g1654/55-215 90/1 897/20-1970/18-19 5 . 0.74 ASP-s0 48.g 07114/2-1/72- 5 ASP-s001 9 0.83 0.76 115/23 30-29 0.86 8 Nam-ASP-g01 159 213 -701 ASP-s004ASP-s0048. Nam-ASP-g01 1369/3-184 048.g1656/229-389 0.81 0.05 0.93 0.36 Nam-ASP-g01364/13-173 1/2 1 0.13 0.38 1 4393/10-181 85 ASP-s0048 7-40 24 0.38 0.88 Nam-ASP-g01363/7-178 8/526 63-3 /7-1 0.81 Nam-ASP-g13 ASP-s011 6 0.95 0.74 8.g1585 6 0.16 Nam-ASP-g0274 .g101 ASP-s0119.g8 g1657/ 8 0.85 0.79 Nam-ASP-g0 .g1019 1 ASP-s00 .g1655/28 0.66 Nam-ASP-g0 023/2-1893-37 3 ASP-s0041 9.g835/ 0.83 0.99 ASP-s0010.g1022/24-189 21 /208-3131-142 0.09 010.g1014/3-187 ASP-s025 0 ASP-s0010 6/21 48.g15 1 ASP 8-458 0.44 ASP-s0010 28/252-405217-36 0.99 .g62 263/43-3/245-408 ASP-s025 ASP-s0 -s0256.g353/273-376.g354/23587/73- 0 0.77 0.97 0.88

6.g35 0.92 . 0.86 ASP-s0010.g1 ASP-s025 8 1 0.94 8 ASP-s0610.g625/257-414 ASP-s0041. 0.88 9 0.64 0.8 59-400 7/256 23 0. 0.87 ASP-s0610 Nam-ASP-g1 6.g364/2 -401 9 0. 0.81 Nam-ASP 5 9 8 ASP-s0017.g3 6 -412 0.82 29-183 6.g349 8 0.9 0.75 ASP-s0017.g326 1 0. ASP- g359/184-29428-326 0.57 0.38 ASP-s0610.g625/54-217 2/30-18 0.91 9-20 -g16753 /5-160 0.98 ASP-s0610.g626/5-174 -215 ASP-ss0119 6755/6 0.93 0.65 5 ASP 0.49 ASP-s0610.g624/2 57/3

ASP- -s093 0119.g810/32-1.g803 0 ASP-s0610.g624/427-587 9

/133-29-241 . 8 0.92 0.76 2 0.87 1 ASP-s0041.g354/ ASP-s0173.gs0216.g5.g /219- 0.16 2 93 0.97 0.74 ASP-s0522.g288 753/3-6 0 ASP-s0173.g394/311 0.84 389 0.94 0 0.52 ASP-s0256.g3 6 ASP-s0151 2381/21/2 1 0.98 0.78 ASP-s0256.g353/112 ASP-s0173.g393 24-3 02 0.22 0.88 395/2 0.92 0.55 0.76 ASP-s0256.g364/18-1264/32-12 7 ASP-s015 35-40 85 0.85 31-13210 .g2841/19 0.92 1 Nam-ASP-g16 7 ASP-s0151.g2847/272-44 37-40 1 1 218-381 1 Nam-ASP-g02290/14-11 0.81 ASP-s0151.g2844/231-391.g2846/23 4 0.98 ASP-s0477.g2175/8-113714/22-/17-17 /218-381-359 0.85 ASP-s0173 0.83 Nam-ASP-g061 .g1585 0. 4 7 0. ASP-s0041.g359/ 0/14-179 ASP-s0048.g1 2-4032 0.28 7 0.61 ASP-s0071.g526/235-397 ASP-s1162.g .g397/248-410 0.35 0.17 ASP-s0048 ASP 3 0.93 55/85-249 ASP -s00 0.99 ASP-s0048.g1591/37-20456/13-180 647/5-7 7 0.4 ASP-s0048.g157 ASP-s0048.g1-s0048.g1605.g2 0.66 0.97 0.24 3 ASP-s0048.g1576/1-11 695/23 3 ASP-s0272.g948/12-1 0.83 0.72 0.9 7 ASP-s0048.g16 03/20-177 9 ASP-s0272. 49/163-32 0. 0. 1/43-201 -183 4 0.74 ASP-s0048.g16 648/2 0.14 0.73 6 ASP-s02 0.19 0. Nam-ASP-g07115/6-179 14-30 3 8 0.94 g933/206-3 ASP-s0119.g8 -132 5 ASP-s0119.g8 8 0.93 0.87 72.g 2 0.07 ASP-s0119.g81 Nam-ASP- 4 2 9

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1 4 3 0046.g1373/177-342 5/140-302 .g776/ 4 - 7-40 -s0005.g241 06/63-17 4-11 - - -g059 g05957/401-536 - -

3-45 - -

------s 2.g6

4 2

s s /79-239 5 s SP-g09589/1-116 9

ASP-s033 1 2-29 8 0 2844/4 46/46-20 2 9 46.g1372/267 /479-645 0

9/28-21 A /

0 0 ASP-s0336.g2889/41-226 49.g1663 B 0 005. 8/24 /221-381 9

0 1 311-472 2 5 117 ASP-s0038 0

5 0 0 0 / 2

/ ASP-s0038.g .g3088/5-150 2 8 3 7 2.g608/ 3 2 /16-18 3 / 0

6 04/ .g615 / 0 0 / / 0

.g2323 7 395/1-15 0 4 5 71/75 05/25-138 1 ASP-s0031.g 5 6 ASP-s0468.g2011/133-31 8 9 4 4 5 3 4 ASP-5C ASP-s0012.g1875/23-160 9 5 . /1430 2-414 2 g2389 1 .

2 g616/2-142 g 1 . . 015.g 5 9 . g /106-19 75-19 0 g g g - 4

g 6 6 ASP-s0002.g 8 6 2 ASP-s0080 ASP-s0080.g1347/10-151 1.g3408/ 4

6.g3072/262-4 . 2764/262-428 1 ASP-s0002.g777/ 1 1 97/242-407 Nam-ASP-g07903/367-5 2

g 2 2 2 35-401 3 6

5 . 7 ASP-s0043.g 7 3 7 1/238-402 /227-3 g

ASP-s0002 g 4 ASP-s04 g 53.g2287 g 0-91 . 9 7

. ASP-s0043.g843/24-201 525.g292 0

7 5 6 0061.g3212/370- 2.g3033/ . . 6

/ -15 6/234-361 142 5 0 6 5 2

0 7 3 7 5 3 3 5 /43-18 0 1

0 /

0 0 1 / 9 ASP-s000 5 / 9 / 2 s0007.g3401/38-141 3 2 2 Nam-ASP-g11184/237- ASP-s0535 0 0 0 s - 4 0 ASP-s0213.g23 0 8 -432 8 5

ASP-s0002 - 3 4 9 2 0

ASPR-s0 0 s 4 ASP-s0053 0 0 -30 92 5

- 9 Nam-ASP-g089 - s 7 3

s s 1

ASP-s 1 - -

- - - - 3

P ASP-s010 4 ASP-s000 ASP-s0 S 8

4 ASP-s0101.g3402 3 4 0

P ASP-s00 ASP- P 3 ASP-s0005.g2 P P 1 S

A 5 ASP-s0101.g3406 2 1

S 7 ASP-s0005.g24 ASP-s0672.g1383/35 S S

A 2 ASP-s023 1

ASP-s0225.g ASP-s0005.g2399/246-4

A A ASP-4C

Figure 4 Domain-based phylogeny of ASP and ASPR genes from A. ceylanicum and N. americanus and ASPR genes from other nematodes. The tree shows a maximum-likelihood phylogeny of protein domains rather than full-length proteins at the tips (as ASP genes sometimes encode two or more tandem ASP domains). All ASP domains and most ASPR domains are from A. ceylanicum or N. americanus. Almost all domains from ASPRs fall within a single branch, labeled in blue. ASPR genes are labeled blue (A. ceylanicum), green (N. americanus), purple (Oesophagostomum dentatum) or magenta (Heligmosomoides bakeri). ASP domains from orthologs of known ASP genes are labeled in gold, with their branches. N- and C-terminal domains from two-domain proteins are noted as “N” or “C.” Domains from other, less familiar ASP genes are labeled in gray. Confidence values are given as decimal fractions (Supplementary Fig. 2). Identities of the corresponding genes and domains are given in Supplementary Tables 4, 13 and 14.

Nature Genetics ADVANCE ONLINE PUBLICATION l e t t e r s also had 92 and 25 ASPR genes, respectively, which were missing with the start of parasitic feeding, molting into L4 larvae and overt entirely from the other species. One explanation for this diversity sexual differentiation (Fig. 1)6,10. We also observed a new protein is the ‘gray pawn’ hypothesis: members of a large gene family might family upregulated at this stage, with homologs in the strongylids have little individual effect on phenotypic fitness yet be collectively A. ceylanicum, N. americanus, H. contortus and Angiostrongylus needed for robust fitness under variable conditions47. For parasites, cantonensis (Supplementary Fig. 3 and Supplementary Tables 4, 12b a relevant variable condition might be diverse host immune systems, and 15); the corresponding genes in A. cantonensis are expressed in which might favor continually diversifying sequences and expression L4 larvae infecting brain tissue48. We thus named this family strong- profiles of ASPs and ASPRs. ylid L4 proteins (SL4Ps). In A. ceylanicum, 24 SL4P genes encoded For development from 24 h to 5 d after infection (24.PI to 5.D), genes proteins of ~200 residues, of which 21 were predicted to be non- encoding structural components of cuticle and genes whose products classically secreted49 without a leader sequence (Supplementary bind cytoskeletal proteins such as actin were prominently upregulated Table 16); notably, parasitic nematodes often use non-classical rather (Supplementary Table 11e). This period in the life cycle corresponds than classical secretion to export proteins into their hosts50.

Haemonchus expansion

Hookworm expansion

Non-parasitic nematodes

Hookworm expansion

Haemonchus expansion

Figure 5 Phylogeny of SCVPs from A. ceylanicum and other nematodes. A maximum-likelihood phylogeny of SCVPs (Supplementary Fig. 5 and Supplementary Tables 4 and 18) is shown. Species are indicated by color: the hookworms A. ceylanicum and N. americanus are shown in green and olive green, respectively; H. contortus is shown in orange; the free-living Caenorhabditis nematodes (C. elegans and C. briggsae) and P. pacificus are shown in blue and light blue; and H. bacteriophora, an insect parasite, is shown in purple. Confidence values are given as decimal fractions (Supplementary Fig. 5b). The SCVP phylogeny falls into five branches: two large, independent gene expansions in hookworms (green); two more branches in H. contortus (orange); and one small branch for non-parasitic nematodes (blue). Like ASPs, SCVPs appear to have existed as a small gene family in free-living nematodes but then to have expanded greatly in both hookworms and other mammalian parasites.

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Table 1 summary of predicted drug targets in A. ceylanicum Protein class A. ceylanicum genes Key C. elegans genes Drug data 4-coumarate:coenzyme A ligase, class I 10 acs-10 NA Ammonium/urea transporter 5 amt-2 NA Cofactor-independent phosphoglycerate mutase 1 ipgm-1 Limited druggability Fumarate reductase 1 F48E8.3 NA Glutamate-gated chloride channel 10 avr-14, avr-15, glc-2 avr-14 observed Glutamate synthase 1 W07E11.1 NA Glutamine-fructose 6-phosphate aminotransferase 3 gfat-1, gfat-2 NA Isocitrate lyase/malate synthase 2 icl-1 NA KH-domain RNA binding 5 asd-2, gld-1, K07H8.9 NA Malate/l-lactate dehydrogenase, YlbC type 4 F36A2.3 NA NADH:flavin oxidoreductase, Oye2/3 type 14 F17A9.4 NA Nematode prostaglandin F synthase 3 C35D10.6 NA O-acetylserine sulfhydrylase 2 cysl-1 NA Secreted lipase 6 lips-8, lips-9 NA Trehalose-6-phosphate synthase 5 gob-1, tps-1, tps-2 gob-1 predicted Predicted drug targets, encoded by 72 genes in A. ceylanicum (Supplementary Table 4), are listed by their protein class. For each class, the number of A. ceylanicum genes encoding it is listed, along with C. elegans homologs that have mutant or RNA interference (RNAi) phenotypes and data indicating whether the drug target is likely to work. avr-14 was recently shown to be a drug target of nitazoxanide67; ipgm-1, previously detected as a promising target, was found to encode a poorly druggable protein68; and gob-1 encodes trehalose-6-phosphatase, a predicted drug target in H. contortus20. “NA” indicates protein classes for which we are not aware of pertinent data. References for all drug target classes (and their drug data, if any) are given in Supplementary Table 19.

From 5 to 12 d after infection (5.D to 12.D), genes encoding parasites but absent from mammals, might be essential for survival protein tyrosine phosphatases, serine/threonine kinases and C-lectins in the host (determined on the basis of C. elegans loss-of-function were prominently upregulated (Supplementary Tables 11g and 12c). phenotypes), had homologs with known three-dimensional protein This period in the life cycle corresponds with maturation from late-L4 structures and had at least one homolog bound by a known small larvae to young adults with incipient fertility and the onset of heavy molecule (Supplementary Fig. 6). This screen yielded 72 genes in blood feeding, which exposes A. ceylanicum to the host’s immune A. ceylanicum, one of which (Acey_s0015.g2804) encoded trehalose- system (Fig. 1)10,11. Among 22 C-lectin genes upregulated by 12 d, 6-phosphatase (Table 1 and Supplementary Tables 4, 19 and 20). we detected 6 whose products had greater apparent similarity to mam- Vaccine targets should be both immunologically accessible and malian than to nematode lectins (Supplementary Tables 4 and 17). crucial for survival. Proteases meet these requirements, as they are Two of these genes encoded structural mimics of mammalian expressed in the intestine (and thus exposed to the host’s immune mannose receptor51, with five tandem C-lectin domains that had system) and because, without them, hookworms cannot digest host arisen through intragenic duplication (Supplementary Fig. 4a). The proteins such as hemoglobin36. We thus selected genes encoding pro- other four C-lectin genes resembled mammalian asialoglycoprotein teases that were permanently upregulated by 5 d after infection and receptors and neurocans51 but arose phylogenetically from nematode that lacked mammalian orthologs but had H. contortus homologs lectins (Supplementary Fig. 4b,c). Lectin genes with similarities to that are also upregulated during infection21. This screen yielded mammalian rather than nematode lectins have also been observed in 12 cathepsin B–like protease genes, with 4 orthologs in H. contortus; the parasitic nematodes Ascaris suum and Toxocara canis and might by 19 d after infection, 5 of these 12 genes generated 1% of all tran- help suppress host immune responses52,53. scripts (Supplementary Table 4). Because protease inhibitors were We also observed a previously undescribed gene family upregulated also upregulated during early infection, we searched for ones meeting at 12 d after infection, with members not only in strongylid parasites our criteria; this screen yielded a previously undescribed protease (A. ceylanicum, N. americanus, H. contortus and Heterorhabditis bacte- inhibitor predicted to be a 79-residue secreted protein with con- riophora) but also in related non-parasitic clade V species (C. elegans, sistently strong expression (~0.1% of all adult transcripts) and one Caenorhabditis briggsae and P. pacificus; Fig. 5, Supplementary Fig. 5 H. contortus homolog upregulated during infection. and Supplementary Tables 12c and 18). We thus named this family The sequencing of A. ceylanicum adds to a growing number of secreted clade V proteins (SCVPs). In A. ceylanicum, 53 SCVP genes genomes for parasitic nematodes that, collectively, infect over encoded ~150-residue proteins, of which 48 were predicted to be clas- 1 billion humans60. Practically, these genomes will be crucial for sically secreted (Supplementary Table 4). Whereas N. americanus and inventing new drugs and vaccines against nematodes that rapidly H. contortus had 11 to 101 SCVP genes, other nematodes had only 1 evolve drug resistance61 and that have been parasitizing vertebrates to 6, suggesting an expansion of SCVP genes in mammalian-parasitic since the Cretaceous62. Understanding immunosuppression by nematodes analogous to those observed for ASP and ASPR genes. parasitic nematodes might also help alleviate autoimmune disorders, A key motivation for parasite genomics is to identify targets for which may be partly due to improved hygiene ridding humans of drugs or vaccines. Because drug development often fails54, it is chronic worm infections63. Intellectually, understanding these genomes essential to identify as many targets as possible. Four drug targets may illuminate remarkable evolutionary changes. Parasitism allows (adenylosuccinate lyase, carnitine O-palmitoyltransferase, dTDP-4- adult nematodes to grow larger and live longer than their free-living dehydrorhamnose 3,5-epimerase and trehalose-6-phosphatase) have relatives (N. americanus adults are ~1 cm long and live for 3–10 years, recently been identified in H. contortus and N. americanus20,24,55–59. whereas C. elegans adults are ~1 mm long and live for 3 weeks), but All four are encoded by genes with A. ceylanicum orthologs the genomic changes underlying these adaptations are essentially (Supplementary Table 4). To identify additional drug targets across unknown1,64–66. The genome and transcriptome of A. ceylanicum the genome, we searched for genes that were conserved by diverse should provide lasting benefits for biology and medicine.

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Nature Genetics ADVANCE ONLINE PUBLICATION ONLINE METHODS Assessing the completeness of genomic DNA. We estimated the assembly’s General summary. Culture and infection of A. ceylanicum in golden hamster completeness as 98% by computing the frequencies of 31-mers71, as 91–99% (Mesocricetus auratus) were carried out as described69. All housing and care by searching for conserved eukaryotic genes (Supplementary Table 3)76 and of laboratory animals used in this study conformed with the US National as 93% by mapping cDNA (assembled independently from RNA-seq reads) Institutes of Health Guide for the Care and Use of Laboratory Animals in to genomic DNA: these calculations supported a consensus value of 95%. Research (see 18-F22) and all requirements and all regulations issued by the The average number of orthologs observed for full-length core eukaryo- US Department of Agriculture (USDA), including regulations implementing tic genes76 was 1.13, which matched averages of 1.11–1.15 in C. elegans, the Animal Welfare Act (Public Law 89-544, US Statutes at Large) as amended C. briggsae and Caenorhabditis tropicalis (all of which are hermaphrodites and (see 18-F23). Stages of A. ceylanicum selected for developmental RNA-seq are thus are completely homozygous), suggesting that the assembly was largely shown in Figure 1 and listed in Supplementary Table 6; they are based on free of unresolved heterozygosity. We searched the genome for tRNA genes previously described stages of growth in golden hamster10. with tRNAscan-SE-1.3.1 (ref. 96); this analysis detected a full complement of Genomic sequencing and RNA-seq were carried out largely as described70. 426 tRNAs decoding all 20 standard amino acids and one selenocysteine tRNA The numbers of A. ceylanicum and hamsters used for A. ceylanicum RNA-seq (Supplementary Table 24). are listed in Supplementary Table 21. The A. ceylanicum genomic sequence was assembled from paired Illumina 100-nt reads (550 nt and 6 kb apart) with Examining repetitive elements for possible horizontal gene transfer. In Velvet (1.2.05)18, gaps were closed after assembly with BGI GapCloser 1.12 A. caninum, the repetitive element bandit resembles the HSMAR1 mariner-like (release_2011)71 and the sequence was reduced in possible heterozygosity72 with transposon of humans and has been postulated to arise from a mammalian HaploMerger (20111230)73. Genomic RNA scaffolding was performed by filter- host by HGT97. To determine whether a bandit homolog also existed in A. cey- ing RNA-seq reads with khmer74 and then scaffolding with ERANGE (3.2)17. lanicum, we searched our library of A. ceylanicum repetitive elements with the RNA-seq reads were assembled into cDNA with Oases (0.2.07)75. Assembled DNA sequence for bandit via BLASTN (2.2.26+)90 (arguments: “-task blastn cDNAs (Supplementary Table 2) were used both to assess genome completeness -evalue 1e-03”). This analysis yielded two hits, with E values of 0.0 and 7 × 10−170 and to aid in the prediction of protein-coding genes. The true genomic size of (Supplementary Table 25). Phylogenetic analysis (Supplementary Fig. 7) and A. ceylanicum was estimated by counting 31-mer frequencies with SOAPdenovo domain analysis with HMMER/hmmsearch indicated that the higher-scoring (V1.05)71, by CEGMA (v2.4.010312) (Supplementary Table 3)76 and by map- hit represented an A. ceylanicum homolog of bandit, whereas the lower-scoring ping cDNAs to genomic DNA with BLAT (v. 34)77. Repetitive DNA elements in hit represented a partial homolog of bandit that did not encode a transposase the final genome assembly were identified with RepeatScout (1.0.5)78. domain (Transposase_1/PF01359.13 in Pfam). We predicted protein-coding genes for our final genomic assembly with To examine whether more evidence for lateral acquisition of repetitive ele- AUGUSTUS (2.6.1)25, after generating species-specific parameters with one ments existed in human hookworms, we used the DFAM database98 to identify round of MAKER2 (2.26-beta)79 (see URLs for the protocol by S. Kumar for repetitive DNA elements in A. ceylanicum and N. americanus with similarity to running MAKER2) and using hints from cDNA that had been mapped to the human repetitive elements. This analysis identified two classes of elements with genome assembly with BLAT. For predicted A. ceylanicum proteins, we annotated mammalian similarities, L3/Plat_L3-like retrotransposons and HSMAR1/2- signal and transmembrane sequences with Phobius80, low-complexity regions like mariner elements (Supplementary Table 25). To determine whether these with SEG81, coiled-coil domains with NCoils82, Pfam 26.0 domains (from both similarities were adventitious or real, we computed maximum-likelihood Pfam-A and Pfam-B)83 with HMMER 3.0/hmmsearch84, InterPro domains with phylogenies for reverse-transcriptase domains (for L3/Plat_L3-like elements) InterProScan (4.8)85 and GO terms with Blast2GO 2.5 (build 23092011)33 (see and transposase domains (for HSMAR1/2-like elements). These phylogenies URLs for protocols for running Blast2GO and InterProScan). We also assigned included all of the L3/Plat_L3-like and HSMAR1/2-like repetitive elements that GO terms to C. elegans and H. contortus genes with Blast2GO so that comparisons we could detect in A. ceylanicum and N. americanus, in a diverse set of other of GO terms between different nematode species would be based on equivalent published genome sequences from nematodes, vertebrates, arthropods, lopho- GO term assignments. We performed InterProScan and Blast2GO for both trochozoans and deuterostomes (Supplementary Table 26) and in a curated A. ceylanicum and C. elegans. We computed orthologies with OrthoMCL (1.3)86. collection of eukaryotic elements from RepBase99 (see URLs for source). We Strict orthologies between genes from two or more species were defined as those extracted well-aligned, full-length protein domains from repetitive elements by orthology groups that contained only one predicted gene for each of the species. requiring that they match the Pfam domains Transposase_1/PF01359.13 (for Annotations for protein-coding genes are listed in Supplementary Table 4. HSMAR1/2-like elements) or RVT_1 (reverse transcriptase)/PF00078.22 (for For RNA-seq analysis of C. elegans, we used published developmental L3/Plat_L3-like elements) and also by excluding the shortest 10% of domain data from the modENCODE consortium (Supplementary Table 22)42. For matches. These criteria led us to select 988 Plat_L3/L3-like RVT_1/PF00078.22 H. contortus, we used published developmental RNA-seq data21. peptides (Supplementary Table 27a) and 168 HSMAR1/2-like Transposase_ We mapped RNA-seq reads to genes with Bowtie 2 (ref. 87) and quantified 1/PF01359.13 peptides (Supplementary Table 27b), which we subjected to gene expression with RSEM (1.2.0)88. For individual genes, we computed multiple-sequence alignment and phylogenetic analysis. the significance for changes in gene activity between stages or biological conditions (Supplementary Tables 7 and 23) with NOISeq-sim (2.13)89. Analyzing protein-coding genes. For motif searches or OrthoMCL analyses Because we had only one biological replicate per condition, we sampled five of protein sequences, we used nematode and mammalian proteomes from random subsets of RNA-seq data per condition to estimate the significance genomic sequences and partial nematode proteomes from translated ESTs. All of changes in gene activity. For A. ceylanicum, C. elegans and H. contortus, we proteomes and their sources are listed in Supplementary Table 28. We classi- used FUNC 0.4.5 with Wilcoxon rank-sum statistics34 to compute which GO fied A. ceylanicum, H. contortus and C. elegans genes both by known protein terms were significantly associated with genes up- or downregulated between motifs (through HMMER 3.0/Pfam-A 26 and InterProScan 4.8)83–85 and evo- developmental stages or environmental conditions (for example, changes lutionary relationships to genes in different species (through OrthoMCL 1.3)86. of drug treatment). For A. ceylanicum, we also used rank-sum statistics to Pfam-A domains were detected at a threshold of E ≤ 1 × 10−5; InterProScan and compute such associations for protein families. OrthoMCL were run with default parameters. We used Pfam-A and InterPro For phylogenetic analyses, sequences homologous to a protein or motifs, in turn, to assign GO terms to each gene with Blast2GO 2.5 (build single domain were extracted with psi-BLAST90 or HMMER/jackhmmer. 23092011)33. We performed InterProScan and Blast2GO according to avail- Protein sequences were aligned with MUSCLE (3.8.31)91 or MAFFT able protocols (see URLs); for Blast2GO, we used both InterProScan predic- (v7.158b)92; alignments were edited with Trimal (v1.4.rev15)93 and visual- tions and BLASTP results against an animal-specific subset of the NCBI nr ized with JalView (2.8)94. Protein maximum-likelihood phylogenies and their database (NCBI-nr)100. We computed orthology groups for our A. ceylanicum branch confidence levels were computed with FastTree (2.1.7)95 and visualized genes with OrthoMCL (1.3)86, for numbers of species ranging from 4 to 14 with FigTree 1.4 (see URLs). (Supplementary Tables 4 and 28). Strict orthologies between genes of two or Some details of these methods are provided below; considerably more more species were defined as those orthology groups that contained only one extensive details are provided in the Supplementary Note. predicted gene for each of those species (Fig. 2). Strict orthologies allowed

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